DESCRIPTION: The long-term objective is to comprehend the relationships between protein conformational change and biological function. Life at its most fundamental level arises from the orchestration of macromolecular movements. To understand protein conformational change is to grasp the underlying principles of cellular biology, human or otherwise. Under physiological conditions, protein conformational change is driven by macromolecular association, the binding of ligands or effectors, changes in pH or oxidation state, electronically excited states, or catalytic chemistry. These mechanisms are often mediated by chromatically active cofactors. Structural information at atomic resolution for experimentally defined protein conformations is extremely powerful for delineating the link between conformation and function. Four proteins: photoactive yellow protein (PYP), Root-effect hemoglobin (Hb), sulfite reductase hemoprotein (SiRHP), and Cu,Zn superoxide dismutase (SOD) represent a wide range of cofactors and biological activities. PYP is a photo-sensing signal-transduction protein that acts through a unique 4-hydroxycinnamic acid chromophore. Time-resolved and low-temperature crystallographic studies will be used to trap photocycle intermediates, whereas biochemical and crystallographic characterization of site-directed mutants will test key structural determinants. Root-effect Hbs exploit extreme pH gradients to pump molecular oxygen into the swim bladder of fish against 1 00 atmospheres of pressure. Characterization and analysis of coupled changes between heme and protein conformation driven by positive-charge clusters at subunit interfaces will reveal the mechanism for this functionally-linked allostery. SIRHP makes use of a cysteine- coupled siroheme-Fe4S4 cluster to catalyze concerted 6-electron reductions of sulfite and nitrite for assimilation of sulfur and nitrogen into the biosphere. Crystallographic structures of SIRHP in its various oxidation states with ligated substrates, intermediates, and products, along with mutagenesis to test hypotheses will provide insight into the reaction mechanism. SODs employ Cu and Zn ions to protect cells from reactive oxygen radicals. Crystallographic and molecular orbital characterization of mutants in different oxidation states, combined with comparisons among species will define essential features for electrostatic recognition of substrate, catalytic metal geometry and a stable subunit fold and dimer interface. These comparisons promise insight into the role of mutant SODs in familial amyotrophic lateral sclerosis (ALS). Overall, the proposed research will contribute to identifying mechanisms of functionally- important conformational change fundamental to many systems of biological and medical interest.